Thermo Fisher Scientific TaqMan Small RNA Assays Mode d'emploi

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Thermo Fisher Scientific
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TaqMan Small RNA Assays
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Mode d'emploi
For Research Use Only. Not for use in diagnostic procedures.
TaqMan™ Small RNA Assays
USER GUIDE
TaqMan™ MicroRNA Assays, Custom TaqMan™ Small RNA
Assays, and TaqMan™ siRNA Assays
Publication Number 4364031
Revision H
Life Technologies Corporation | 6055 Sunol Blvd | Pleasanton, CA 94566
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Revision history: Pub. No. 4364031
Revision Date Description
H 10 December 2019 Removed troubleshooting and replaced with link to FAQs.
G 23 July 2019 • Corrected volumes for PCR Reaction Mix and amount of cDNA
template to add.
• Added QuantStudio™ 6 Pro Real-Time PCR System and
QuantStudio™ 7 Pro Real-Time PCR System.
• Removed the instruction to add a compression pad to the real-time
PCR plate.
• Added a recommended starting point for the threshold value when
analyzing the data.
• Added information about UNG.
• Added information about secondary analysis software.
• Removed recommendation to use the Megaplex™ Assay
Performance File in troubleshooting non-specific interactions
between primers.
F 12 February 2019 • Added new instruments, Master Mixes, and other applicable
products.
• Added thermal cycling protocols for all compatible Master Mixes.
• Updated options for secondary analysis software.
• Added troubleshooting information.
• Updated for general style, formatting, and branding.
E January 2011 Baseline for this revision history.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered
trademark of Roche Molecular Systems, Inc., used under permission and license. AmpErase is a trademark of Roche Molecular Systems, Inc.
Eppendorf and MixMate are trademarks of Eppendorf AG.
©2019 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■CHAPTER 1 Product information ....................................... 5
Product description ............................................................. 5
Overview of TaqMan™ Small RNA Assays ........................................... 6
TaqMan™ Small RNA Controls .................................................... 6
Contents and storage ............................................................ 7
Order TaqMan™ Small RNA Assays ............................................ 8
Required materials not supplied .................................................. 8
Workflow ..................................................................... 11
■CHAPTER 2 Perform reverse transcription .......................... 12
Guidelines for isolation of high–quality RNA ....................................... 12
Guidelines for RNA input ........................................................ 12
Before you begin ............................................................... 13
Prepare the RT Reaction Mix .................................................... 13
Prepare the RT reaction ......................................................... 14
Prepare RT reaction with double–stranded small RNA .......................... 14
Prepare RT reaction with single–stranded small RNA .......................... 14
Perform reverse transcription ................................................... 15
■CHAPTER 3 Perform PCR amplification .............................. 16
Procedural guidelines for performing real–time PCR ............................... 16
Before you begin ............................................................... 16
Prepare the PCR Reaction Mix ................................................... 17
Prepare the PCR reaction plate .................................................. 17
Set up and run the real–time PCR instrument ...................................... 18
Analyze the results ............................................................. 19
Secondary analysis software ................................................ 20
Algorithms for data analysis ................................................ 20
TaqMan
™
Small RNA Assay User Guide
3
■APPENDIX A Troubleshooting and FAQs .............................. 21
■APPENDIX B Supplemental information .............................. 22
Overview of TaqMan™ Small RNA Assay chemistry ................................. 22
Reverse transcription ...................................................... 22
TaqMan™ MGB probes ...................................................... 23
About the 5' nuclease assay ................................................. 23
Best practices for PCR and RT-PCR experiments ................................... 25
Good laboratory practices for PCR and RT-PCR ................................ 25
Use UNG to prevent false-positive amplification ............................... 25
Detect fluorescent contaminants ............................................ 25
■APPENDIX C Safety ..................................................... 26
Chemical safety ................................................................ 27
Biological hazard safety ......................................................... 29
■Documentation and support ............................................. 30
Related documentation ......................................................... 30
Customer and technical support ................................................. 30
Limited product warranty ....................................................... 30
Contents
4
TaqMan
™
Small RNA Assay User Guide
Product information
■Product description .................................................... 5
■Overview of TaqMan™ Small RNA Assays ............................... 6
■TaqMan™ Small RNA Controls .......................................... 6
■Contents and storage .................................................. 7
■Required materials not supplied ......................................... 8
■Workow ........................................................... 11
Product description
Applied Biosystems™ TaqMan™ Small RNA Assays are primer and probe sets
designed to detect and quantify mature microRNAs (miRNAs), small interfering
RNAs (siRNAs), and other small RNAs. The assays can detect and quantify small
RNA in 1 to 10 ng of total RNA with a dynamic range of greater than six logs. When
used for microRNA analysis, the assays can discriminate mature miRNA sequences
from their precursors.
Note: In this user guide, “small RNA” refers to miRNA, siRNA, or other small RNAs
that are less than 200 bases in length.
Predesigned and custom TaqMan™ Small RNA Assays are available for a variety of
small RNA classes.
• TaqMan™ MicroRNA Assays
– Predesigned assays for the majority of content found in the miRBase miRNA
sequence repository.
– Ideal for targeted quantication, screening, and validation of miRNA
proling results.
• TaqMan™ siRNA Assays for Silencer™ Select siRNAs
– Predesigned assays for the quantication of Silencer™ Select siRNAs.
– Ideal for assessing siRNA transfection eciency, half-life, and bio-
distribution.
• TaqMan™ Small RNA Controls
– Predesigned assays for small, non-coding RNAs unrelated to miRNAs used
to normalize for dierences in sample RNA.
– TaqMan™ Small RNA Controls are available for a number of species.
For more information on using TaqMan™ control assays, see “Select a TaqMan™
Small RNA Control” on page 6.
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TaqMan
™
Small RNA Assay User Guide
5
• Custom TaqMan™ Small RNA Assays
– Custom assays designed for any small RNA sequence from
17–200 nucleotides in length.
– We provide ready-to-use Custom TaqMan™ Small RNA Assays based on
customer–supplied target sequences for any organism, with optimized
primers and probe.
Overview of TaqMan™ Small RNA Assays
TaqMan™ Small RNA Assays use a stem-looped primer for reverse transcription and a
sequence-specic assay to accurately detect mature miRNAs, siRNAs, and other small
RNAs.
Each assay includes two tubes.
• A tube of small RNA–specic stem–looped RT Primer.
• A tube containing a mix of small RNA–specic forward PCR Primer, small RNA–
specic reverse PCR Primer, and small RNA–specic TaqMan™ MGB probe.
For a current list of available assays, use the assay search tool at thermosher.com/
taqmanmirna.
TaqMan™ Small RNA Controls
We recommend using TaqMan™ Small RNA Controls with TaqMan™ Small RNA
Assays. When quantifying small RNA gene expression levels, variation in the amount
of starting material, sample collection, RNA preparation and quality, and reverse
transcription (RT) eciency can contribute to quantication errors. Normalization to
endogenous control genes is currently the most accurate method to correct for
potential RNA input or RT eciency biases.
An ideal endogenous control generally shows gene expression that is relatively
constant and highly abundant across tissues and cell types. You need to verify the
chosen endogenous control or set of controls for the target cell, tissue, or treatment
because no single control can act as a universal endogenous control for all
experimental conditions.
To view a complete list of available controls, use the assay search tool at
thermosher.com/taqmanmirna.
Chapter 1 Product information
Overview of TaqMan
™
Small RNA Assays
1
6
TaqMan
™
Small RNA Assay User Guide
Contents and storage
Table 1 TaqMan™ MicroRNA Assays
Cat. No. Number of
20–µL reactions
Amount and concentration
Storage
RT Primer Assay
Inventoried predesigned assays
4427975
(Small)
50 RT
150 PCR 150 µL (5✕) 150 µL (20✕) −25°C to −15°C
Made–to–order predesigned assays
4440888
(Large)
2900 RT
2900 PCR 725 µL (60✕) 967 µL (60✕)
−25°C to −15°C
4440887
(Medium)
750 RT
750 PCR 575 µL (20✕) 750 µL (20✕)
4440886
(Small)
50 RT
150 PCR 150 µL (5✕) 150 µL (20✕)
4440885
(Extra small)
25 RT
75 PCR 75 µL (5✕) 75 µL (20✕)
Table 2 Custom TaqMan™ Small RNA Assays
Cat. No. Number of
20–µL reactions
Amount and concentration
Storage
RT Primer Assay
4398989
(Large)
2900 RT
2900 PCR 725 µL (60✕) 967µL (60✕)
−25°C to −15°C
4398988
(Medium)
750 RT
750 PCR 575 µL (20✕) 750 µL (20✕)
4398987
(Small)
50 RT
150 PCR 150 µL (5✕) 150 µL (20✕)
4440418
(Extra small)
25 RT
75 PCR 75 µL (5✕) 75 µL (20✕)
Chapter 1 Product information
Contents and storage
1
TaqMan
™
Small RNA Assay User Guide
7
Table 3 TaqMan™ siRNA Assays (predesigned)
Cat. No. Number of
20–µL reactions
Amount and concentration
Storage
RT Primer Assay
4440880
(Large)
2900 RT
2900 PCR 725 µL (60✕) 967 µL (60✕)
−25°C to −15°C
4440879
(Medium)
750 RT
750 PCR 575 µL (20✕) 750 µL (20✕)
4440878
(Small)
50 RT
150 PCR 150 µL (5✕) 150 µL (20✕)
4440877
(Extra small)
25 RT
75 PCR 75 µL (5✕) 75 µL (20✕)
Order predesigned and custom assays for miRNAs, siRNAs, and other small RNAs at
thermosher.com/taqmanmirna. There are tools to help select inventoried assays or
design custom assays for an unlisted small RNA.
Order Custom TaqMan™ Small RNA Assays or predesigned TaqMan™ siRNA Assys
for Silencer™ Select siRNAs at Silencer Select siRNAs.
For information on designing custom assays, see Custom TaqMan™ Small RNA Assays
Design and Ordering Guide (Pub. No. 4412550).
Required materials not supplied
Unless otherwise indicated, all materials are available through thermosher.com.
MLS: Fisher Scientic (sherscientic.com) or other major laboratory supplier.
Table 4 Recommended products for isolation of RNA
Sample type Item Source
Tissue
samples
mir
Vana™ miRNA Isolation Kit, with phenol AM1560
mir
Vana™ miRNA Isolation Kit, without phenol AM1561
mir
Vana™ PARIS™ RNA and Native Protein Purification
Kit AM1556
MagMAX™
mir
Vana™ Total RNA Isolation Kit A27828
TRI Reagent™ Solution AM9738
RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975
Cell samples
TaqMan™ MicroRNA Cells-to-CT Kit 4391848
Cells-to-CT
™ Stop Solution, 1 mL 4402960
Cells-to-CT
™ Bulk Lysis Reagents 4391851C
Order TaqMan™
Small RNA Assays
Chapter 1 Product information
Required materials not supplied
1
8
TaqMan
™
Small RNA Assay User Guide
Sample type Item Source
Cell samples MagMAX™
mir
Vana™ Total RNA Isolation Kit A27828
Liquid
samples MagMAX™
mir
Vana™ Total RNA Isolation Kit A27828
Table 5 Recommended product for preparation of cDNA
Item Source
TaqMan™ MicroRNA Reverse Transcription Kit[1] 4366596
[1] TaqMan™ Small RNA Assays are optimized for the MuLV Reverse Transcriptase contained in the TaqMan™
MicroRNA Reverse Transcription Kit. Performance with other RT enzymes cannot be guaranteed.
Table 6 PCR Master Mixes
Item Source
TaqMan™ Fast Advanced Master Mix 4444558
TaqMan™ Universal Master Mix II, no UNG 4440043
TaqMan™ Universal Master Mix II, with UNG 4440042
TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG 4364341
TaqMan™ Universal PCR Master Mix 4304437
Table 7 Other materials and equipment required for the workflow
Item Source
Real-time PCR instrument, one of the following:
QuantStudio™ 6 Pro and 7 Pro Real-Time PCR Systems
Contact your local
sales office
QuantStudio™ 3 or 5 Real-Time PCR System
QuantStudio™ 6 / QuantStudio™ 7 Flex Real-Time PCR System
QuantStudio™ 12K Flex Real–Time PCR System
StepOne™ or StepOnePlus™ Real-Time PCR System
ViiA™ 7 Real-Time PCR System
7500/7500 Fast Real-Time PCR System
Equipment
Thermal cycler, one of the following (or equivalent):
• Veriti™ Thermal Cycler
• SimpliAmp™ Thermal Cycler
• ProFlex™ PCR System
Contact your local
sales office
Centrifuge, with adapter for 96-well or 384-well plates MLS
Chapter 1 Product information
Required materials not supplied
1
TaqMan
™
Small RNA Assay User Guide
9
Item Source
Microcentrifuge MLS
Vortex mixer MLS
(Optional)
Eppendorf™ MixMate™ (shaker) Fisher Scientific™
21-379-00
Pipettes MLS
Tubes, plates, and other consumables
Tubes, plates, and film thermofisher.com/
plastics
Aerosol-resistant barrier pipette tips MLS
Disposable gloves MLS
Reagents
Nuclease-free Water AM9930
RNase Inhibitor N8080119
RNaseOUT™ Recombinant Ribonuclease Inhibitor 10777019
TURBO DNA-
free
™ KitDNase AM1907
TE, pH 8.0 AM9849
Chapter 1 Product information
Required materials not supplied
1
10
TaqMan
™
Small RNA Assay User Guide
Workflow
Start with total RNA
▼
Prepare the RT Reaction Mix (page 13)
▼ ▼
Prepare RT reaction with double–
stranded small RNA (page 14)
Prepare RT reaction with single–
stranded small RNA (page 14)
▼
Perform reverse transcription (page 15)
▼
Prepare the PCR Reaction Mix (page 17)
▼
Prepare the PCR reaction plate (page 17)
▼
Set up and run the real–time PCR instrument (page 18)
▼
Analyze the results (page 19)
Chapter 1 Product information
Workflow
1
TaqMan
™
Small RNA Assay User Guide
11
Perform reverse transcription
■Guidelines for isolation of high–quality RNA ............................ 12
■Guidelines for RNA input ............................................. 12
■Before you begin ..................................................... 13
■Prepare the RT Reaction Mix ........................................... 13
■Prepare the RT reaction ............................................... 14
■Perform reverse transcription .......................................... 15
Guidelines for isolation of high–quality RNA
• See Table 4 on page 8 for recommended RNA isolation kits.
• Prepare samples using a method that preserves small RNAs.
• To prevent the loss of the longer control transcripts (such as snoRNAs), size
fractionation is not recommended.
Guidelines for RNA input
• Use 1–10 ng of total RNA per 15–µL RT reaction.
• For optimal reverse transcription, input RNA should have the following
characteristics:
– Free of inhibitors of reverse transcription (RT) and PCR.
– Dissolved in PCR-compatible buer.
– Free of RNase activity.
– Nondenatured total RNA (not applicable for double-stranded templates).
IMPORTANT! Do not denature the total RNA.
• Use 10 to 10,000 cells per sample for the TaqMan™ MicroRNA Cells-to-CT Kit.
• When working with double-stranded template such as siRNAs and miRNA
mimics, denature the siRNA template with sequence-specic RT Primer before
performing the reverse transcription.
• Prepare the RT reactions in an area free of articial templates, amplied material,
and siRNA transfections. High-copy-number templates can easily contaminate
the reactions.
• When working with siRNA use total RNA from non-transfected cells as a control
in in vitro and in vivo studies. The amount of input total RNA for optimal
detection depends on the transfection protocol used.
2
12
TaqMan
™
Small RNA Assay User Guide
Note: When testing for more than 10 targets, it might be more ecient to create
primer pools. For details see Protocol for Creating Custom RT and Preamplication Pools
Using TaqMan™ MicroRNA Assays (Pub. No. 4465407).
IMPORTANT! TaqMan™ MicroRNA Assays and TaqMan™ Small RNA Assays are
optimized for the MuLV Reverse Transcriptase contained in the TaqMan™ MicroRNA
Reverse Transcription Kit. Assay performance with other reverse transcriptase
enzymes cannot be guaranteed.
Before you begin
• Thaw components of the reverse transcription kit on ice.
• Thaw the RT Primers on ice, vortex briey, then centrifuge briey to collect the
contents at the boom of the tube.
• (Large assays only) Dilute the 20✕ or 60✕ RT Primer to a 5✕ working solution
using 0.1✕ TE Buer. Store for up to one year at −25°C to −15°C.
Prepare the RT Reaction Mix
1. In an appropriately-sized microcentrifuge tube, prepare RT Reaction Mix
according to the following table.
Component Volume
(1 reaction)
Volume
(10 reactions)[1]
100mM dNTPs (with dTTP) 0.15 µL 1.65 µL
MultiScribe™ Reverse
Transcriptase, 50 U/µL 1.00 µL 11.00 µL
10✕ Reverse Transcription Buffer 1.50 µL 16.50 µL
RNase Inhibitor, 20 U/µL 0.19 µL 2.09 µL
Nuclease-free Water 4.16 µL 45.76 µL
Total RT Reaction Mix volume 7.00 µL 77.00 µL
[1] Includes 10% overage.
2. Invert to mix, then centrifuge briey to collect the contents at the boom of the
tube.
Place the RT Reaction Mix on ice. Proceed immediately to “Prepare the RT
reaction“ on page 14.
Chapter 2 Perform reverse transcription
Before you begin
2
TaqMan
™
Small RNA Assay User Guide
13
Prepare the RT reaction
Use one of the following procedures.
Silencer Select miRNAs are double–stranded miRNA–mimicking molecules.
1. Combine 3 µL of 5✕ RT Primer and 5 µL of double-stranded template in a
reaction tube or in each well of a reaction plate.
5 µL should contain 1–10 ng of double–stranded template.
2. Mix thoroughly, then centrifuge briey to collect the contents at the boom of the
tube or wells.
3. Incubate at 85°C for 5 minutes.
4. Incubate at 60°C for 5 minutes, then place on ice.
5. Add 7 µL of RT Reaction Mix to each reaction tube or well.
RT Reaction Mix was prepared in “Prepare the PCR Reaction Mix“ on page 17.
6. Seal the tube or reaction plate.
7. Centrifuge briey to collect the contents at the boom of the tubes or wells.
Place on ice and proceed immediately to “Perform reverse transcription“ on page 15.
1. Combine 7 µL of RT Reaction Mix and 5 µL of total RNA in a reaction tube or in
each well of a reaction plate.
5 µL should contain 1–10 ng of total RNA.
RT Reaction Mix was prepared in “Prepare the RT Reaction Mix“ on page 13.
2. Mix thoroughly, then centrifuge briey to collect the contents at the boom of the
tubes or wells.
3. Add 3 µL of 5✕ RT Primer to each reaction tube or each well of a reaction plate.
4. Seal the tubes or reaction plate.
5. Centrifuge briey to collect the contents at the boom of the tubes or wells.
Place on ice and proceed immediately to “Perform reverse transcription“ on page 15.
Prepare RT
reaction with
double–stranded
small RNA
Prepare RT
reaction with
single–stranded
small RNA
Chapter 2 Perform reverse transcription
Prepare the RT reaction
2
14
TaqMan
™
Small RNA Assay User Guide
Perform reverse transcription
Place the reaction plate or tubes into a thermal cycler, then incubate using standard
cycling, a reaction volume of 15.0 µL, and the following seings.
Step Temperature Time
Reverse transcription 16°C 30 minutes
42°C 30 minutes
Stop reaction 85°C 5 minutes
Hold 4°C Hold
The RT reaction product can be stored at –25 to –15°C for up to one week.
Chapter 2 Perform reverse transcription
Perform reverse transcription
2
TaqMan
™
Small RNA Assay User Guide
15
Perform PCR amplification
■Procedural guidelines for performing real–time PCR ...................... 16
■Before you begin ..................................................... 16
■Prepare the PCR Reaction Mix ......................................... 17
■Prepare the PCR reaction plate ......................................... 17
■Set up and run the real–time PCR instrument ............................ 18
■Analyze the results ................................................... 19
Procedural guidelines for performing real–time PCR
• Follow best practices when preparing or performing PCR. See “Good laboratory
practices for PCR and RT-PCR“ on page 25.
• Prepare the PCR reactions in an area free of articial templates and siRNA
transfections. High-copy-number templates can easily contaminate the real-time
PCR reactions.
• Protect the assays from light and store in a freezer. Excessive exposure to light
might aect the uorescent probes.
• Prepare the PCR reaction mix before transferring it to the reaction plate for
thermal cycling and uorescence analysis.
• Perform three replicates of each reaction.
• Include the following reactions:
– A small RNA assay for each cDNA sample.
– Endogenous control assays for each cDNA sample.
– No–template controls (NTCs) for each assay on the plate.
Note: Use of NTC reactions to evaluate background signal is strongly recommended.
Before you begin
• (For large assays) Dilute 60✕ assays to 20✕ working solutions before use.
• Thaw assays (20✕) and cDNA templates on ice, vortex gently, then centrifuge
briey to bring the contents to the boom of the tube.
3
16
TaqMan
™
Small RNA Assay User Guide
Prepare the PCR Reaction Mix
1. Mix the PCR Master Mix by gently swirling the bole.
See “Required materials not supplied“ on page 8 for a list of compatible PCR
Master Mixes.
2. Prepare the PCR Reaction Mix in appropriately-sized microcentrifuge tubes,
according to the following table.
Component
Volume per reaction[1]
384–well or 96–well fast
(0.1mL) plate
96–well standard (0.2 mL)
plate
TaqMan™ Small RNA Assay
(20X) 0.50 µL 1.00 µL
PCR Master Mix 5.00 µL 10.00 µL
Nuclease–free water 3.84 µL 7.67 µL
Total PCR Reaction Mix
volume 9.34 µL 18.67 µL
[1] Add 10% overage to account for pipetting loss.
Note: The volume of nuclease-free water can be adjusted if a smaller volume of
cDNA template is added. The recommended volume of cDNA template is the
maximum volume of cDNA template that should be added, due to the required
dilution of the RT Primer in the nal PCR (see “Prepare the PCR reaction
plate“ on page 17).
3. Vortex to mix the PCR Reaction Mix thoroughly, then centrifuge briey to collect
the contents at the boom of the tube.
Prepare the PCR reaction plate
1. Transfer the appropriate volume of PCR Reaction Mix to each well of an optical
reaction plate.
• 384–well or 96–well fast (0.1 mL) plate: 9.34 µL
• 96–well standard (0.2 mL) plate: 18.67µL
The PCR Reaction Mix was prepared in “Prepare the PCR Reaction Mix“ on
page 17.
2. Add cDNA template, or nuclease–free water for NTC, to each well of an optical
reaction plate.
• 384–well or 96–well fast (0.1 mL) plate: 0.67 µL
• 96–well standard (0.2 mL) plate: 1.33 µL
Note: This is the maximum volume of cDNA template that can be added to the
PCR reaction.
Chapter 3 Perform PCR amplification
Prepare the PCR Reaction Mix
3
TaqMan
™
Small RNA Assay User Guide
17
Adjust the volume of nuclease–free water in the PCR Reaction Mix for a smaller
volume of cDNA template (see “Prepare the PCR Reaction Mix“ on page 17).
3. Seal the plate with optical adhesive lm, then centrifuge the plate briey to bring
the PCR Reaction Mix to the booms of the wells.
Set up and run the real–time PCR instrument
See the appropriate instrument user guide for detailed instructions to program the
thermal-cycling conditions or to run the plate.
Note: The instrument must be congured with the block appropriate for the plate
type.
1. Select the cycling mode appropriate for the Master Mix.
IMPORTANT! The cycling mode depends on the Master Mix that is used in the
reaction. The cycling mode does not depend on a Standard or a Fast plate format.
2. Set up the thermal protocol for your instrument.
Table 8 TaqMan™ Fast Advanced Master Mix (StepOne™, StepOnePlus™, ViiA™ 7,
and QuantStudio™ systems with fast cycling mode)
Step Temperature Time Cycles
(
Optional
) UNG activation 50°C 2 minutes 1
Enzyme activation 95°C 20 seconds 1
Denature 95°C 1 second 40
Anneal / Extend 60°C 20 seconds
Table 9 TaqMan™ Fast Advanced Master Mix (7500 and 7500 Fast systems with
fast cycling mode)
Step Temperature Time Cycles
(
Optional
) UNG activation 50°C 2 minutes 1
Enzyme activation 95°C 20 seconds 1
Denature 95°C 3 seconds 40
Anneal / Extend 60°C 30 seconds
Chapter 3 Perform PCR amplification
Set up and run the real–time PCR instrument
3
18
TaqMan
™
Small RNA Assay User Guide
Table 10 TaqMan™ Universal Master Mix II, no UNG, TaqMan™ Universal Master
Mix II, with UNG, TaqMan™ Universal PCR Master Mix, no AmpErase™ UNG, or
TaqMan™ Universal PCR Master Mix (any compatible instrument with standard
cycling mode)
Step Temperature Time Cycles
(
Optional
) UNG activation 50°C 2 minutes 1
Enzyme activation 95°C 10 minutes 1
Denature 95°C 15 seconds 40
Anneal / Extend 60°C 60 seconds
3. Set the appropriate reaction volume.
4. Load the plate into the real–time PCR instrument.
5. Start the run.
Analyze the results
For detailed information about data analysis, see the appropriate documentation for
your instrument. Use the absolute or relative quantication (ΔΔCt) methods to
analyze results.
The general guidelines for analysis include:
• View the amplication plot; then, if needed:
– Adjust the baseline and threshold values.
A threshold value of 0.2 is a recommended starting point.
– Remove outliers from the analysis.
• In the well table or results table, view the Ct values for each well and for each
replicate group.
Perform additional analysis using any of the following software:
Software Resource
Relative Quantification application
thermofisher.com/cloud
Standard Curve application
ExpressionSuite™ Software[1] thermofisher.com/expressionsuite
DataAssist™ Software thermofisher.com/dataassist
[1] Can automatically define the baseline.
For more information about real-time PCR, see Introduction to Gene Expression Geing
Started Guide (Pub. No. 4454239) or go to thermosher.com/qpcreducation.
Chapter 3 Perform PCR amplification
Analyze the results
3
TaqMan
™
Small RNA Assay User Guide
19
ExpressionSuite™ Software utilizes the comparative CT (ΔΔCT) method to quantify
relative gene expression across a large number of samples.
ExpressionSuite™ Software is available for download at thermosher.com/
expressionsuite.
DataAssist™ Software is a data analysis tool for sample comparison when using the
comparative CT (ΔΔCT) method for calculating relative quantitation of gene
expression. The software uses a ltering procedure for outlier removal and various
normalization methods based on lists of single or multiple genes. It provides relative
quantication analysis of gene expression through a combination of statistical analysis
and interactive visualization.
DataAssist™ Software is available for download at thermosher.com/dataassist.
Table 11 Algorithm recommendations for single–tube assays
Algorithm Recommendation
Threshold (Ct) Recommended.
Relative threshold (Crt)
(Optional)
Use for troubleshooting abnormal or unexpected
results.
The relative threshold algorithm is available in the Relative Quantication application
on Connect (thermosher.com/connect).
Secondary
analysis software
Algorithms for
data analysis
Chapter 3 Perform PCR amplification
Analyze the results
3
20
TaqMan
™
Small RNA Assay User Guide
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Thermo Fisher Scientific TaqMan Small RNA Assays Mode d'emploi

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Thermo Fisher Scientific
Modèle
TaqMan Small RNA Assays
Taper
Mode d'emploi
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